This Mentored Career Development Award (K08) proposal describes a five year training program for the physician scientist candidate with a long-term goal of becoming an academic authority in cellular and molecular mechanosensitivity in the gastrointestinal (GI) tract in health and disease. The candidate acquired a strong basic science foundation in ion channel biophysics during his PhD and post-doctoral work. The candidate now proposes to solidify his foundation and acquire a set of novel complementary skills necessary for an independent research career in GI tract mechanosensitivity. Mechanosensitivity is important for normal GI tract function and abnormal mechanosensitivity leads to disease. Enterochromaffin (EC) cells are mechanosensitive GI epithelial cells that synthesize and release serotonin (5-HT), which regulates GI motility and sensation. Mechanical forces are a major stimulus for 5-HT release by the EC cell, but the molecular mechanisms of EC cell mechanosensation are unclear. Acute cellular mechanosensation involves mechanosensitive ion channels (MSCs). A recently cloned MSC PIEZO2 is critical for Merkel cell mechanosensation, which is a mechanosensitive cell of the skin epithelium with developmental and functional similarities to the EC cell. The central hypothesis of this application is that PIEZO2 ion channels are the primary mechanosensors that transduce mechanical energy into 5-HT release by the EC cells. We will test the central hypothesis in 2 AIMs. The experiments in AIM 1 will determine the localization and mechanical activation of PIEZO2 in EC cells. The experiments in AIM 2 will determine how PIEZO2 activation is coupled to 5-HT release in single EC cells and intestinal organoids and how PIEZO2 activation couples to GI motility. The AIMs are supported by strong preliminary data which show that PIEZO2 is specific to human and mouse colon EC cells, that mechanical stimuli activate PIEZO2 in primary EC cells, leading to release of 5-HT, while the block of PIEZO2 in the colon epithelium decreases motility. To test the central hypothesis, we will use immunohistochemistry and super resolution imaging, electrophysiology and optogenetics in parallel with mechanostimulation of single cells and intestinal organoids, 5-HT measurements by electrochemistry and novel techniques, novel ex vivo and in vivo recordings of colonic motility in wild type and transgenic animals. This work will be performed in an academically nurturing environment within Mayo Clinic and with full support of the Division of Gastroenterology & Hepatology. The candidate will be supported by several consultants who are experts in the areas that the candidate identified as necessary training. He will be guided by a strong mentorship committee (Drs. Gianrico Farrugia and Nicholas LaRusso). Both are international authorities - Dr. Farrugia in ion channels in GI motility, and Dr. LaRusso in epithelial biology and mechanosensation. As a result of this work, the candidate will significantly advance our understanding of EC cell mechanosensitivity mechanisms in human physiology with a goal of providing a novel platform for therapeutic strategies as well as obtain data for a R01 application.